Atrial natriuretic peptide (ANP) is a hypotensive hormone that regulates sodium excretion, fluid volume, and vasorelaxation, important factors in the control of blood pressure and blood volume. One of the principal loci involved in the regulatory action of ANP is the guanylyl cyclase-linked ANP receptor (GC-A) designated as NPRA that belongs to a family of GC-receptors for which collectively the signaling mechanisms are not well understood. NPRA consists of a single polypeptide containing an extracellular ANP-binding domain, a single transmembrane sequence, and intracellular protein kinase-like homology domain (protein-KHD) and guanylyl cyclase catalytic domain. It has been postulated that the binding of ANP to the extracellular domain causes a conformational change, transmitting the signal to cyclase catalytic active center. It has also been proposed that ATP, a positive allosteric effector, binds to the protein-KHD and, hence, augments ANP-dependent guanylyl cyclase activation, however, further studies are needed to reveal the actual mechanisms of signal transduction. The long-term goals of the proposed studies are to elucidate the structure-function relationship of NPRA and to understand how ANP/ATP interact with the receptor and how such interactions are transduced to the intracellular domain to stimulate GC activity and cGMP production, receptor internalization, trafficking, and ultimately inactivation and degradation in human embryonic kidney 293 cells stably expressing recombinant NPRA. Four specific aims are proposed: 1) examine the mechanisms that modulate ANP/ATP signal transduction activities and reveal the structural motifs defining the identity of ATP-binding sequence and activation of NPRA, 2) delineate the molecular mechanisms of the dynamics of internalization, trafficking, and sequestration of NPRA-tagged with green fluorescent protein, 3) examine the molecular mechanisms of ANP action that can be regulated at the level of NPRA by reducing the number of active receptors (down-regulation) or by functional inactivation (desensitization) of constant receptors, and 4) delineate the molecular mechanisms of ANP-mediated physiological functions of NPRA in vascular smooth muscle and mesangial cells transiently expressing wild-type and mutant receptors. The proposed studies should provide a comprehensive assessment of the mode of functioning of NPRA to directly elucidate the unique molecular, pharmacologic and physiologic implications of NPRA signaling pathways at the molecular level. The resulting knowledge should yield new therapeutic targets and novel loci for the control and treatment of hypertension and cardiovascular disorders